Biocatalysis reproduce nature’s synthetic strategies in order to synthesize different organiccompounds. Natural metabolic pathways usually involve complex networks to support cellulargrowth and survival. In this regard, multi-enzymatic systems are valuable tools for the production ofa wide variety of organic compounds. Methods: The production of different purine nucleosides andnucleoside-5′-monophosphates has been performed for first time, catalyzed by the sequential action of2′-deoxyribosyltransferase fromLactobacillus delbrueckii(LdNDT) and hypoxanthine-guanine-xanthinephosphoribosyltransferase fromThermus themophilusHB8 (TtHGXPRT). Results: The biochemicalcharacterization ofLdNDT reveals that the enzyme is active and stable in a broad range ofpH, temperature, and ionic strength. Substrate specificity studies showed a high promiscuityin the recognition of purine analogues. Finally, the enzymatic production of different purinederivatives was performed to evaluate the efficiency of multi-enzymatic systemLdNDT/TtHGXPRT.Conclusions: The production of different therapeutic purine nucleosides was efficiently catalyzed byLdNDT/TtHGXPRT. In addition, the resulting by-products were converted to IMP and GMP. Takingall of these features, this bioprocess entails an efficient, sustainable, and economical alternative tochemical synthetic methods